Stable intravenously injectable plasma protein free from hypotensive effects and process for its production

ABSTRACT

The depressor effect observed when heat-treated plasma protein fractions are rapidly infused is eliminated by separating, e.g., with a surface active adsorbent or cation exchanger, ultrafiltration membrane or by gel molecular sieve, a low molecular weight depressor substance having a molecular weight of less than 10,000 from the plasma proteins.

United States Patent [191 Izaka et al.

Apr. 8, 1975 STABLE INTRAVENOUSLY INJECTABLE PLASMA PROTEIN FREE FROMHYPOTENSIVE EFFECTS AND PROCESS FOR ITS PRODUCTION Inventors: Kenichilzaka; Eizo Tsutsui. both of Osaka Prefecture, Japan Assignee: CutterLaboratories. Inc., Berkeley.

Calif.

Filed: June 13, 1973 Appl. No.: 369,478

Foreign Application Priority Data June 19. 1972 Japan 47-061210 US. Cl.424/177 Int. Cl. 1. C07c 103/52 Field of Search 424/177 [56] ReferencesCited UNITED STATES PATENTS 3.472.831 10/1969 Vester et a1. 424/1773.492.212 1/1970 Searcy 424/177 3.717.708 2/1973 Wada et a1 4 424/177Primary ExaminerElbert L. Roberts Attorney. Agent. or Firm-Millen,Raptes & White [57] ABSTRACT 27 Claims, No Drawings STABLE INTRAVENOUSLYINJECTABLE PLASMA PROTEIN FREE FROM HYPOTENSIVE EFFECTS AND PROCESS FORITS PRODUCTION BACKGROUND OF THE INVENTION This invention relates tostable plasma proteins, substantially free of blood pressure depressantcomponents, and to a method for producing such depressantfree plasmaproteins.

Solutions of heat-treated human plasma protein fractions, e.g., thosedescribed in US. Pat. No. 2,958,628, have been used extensively for anumber of years in clinics and hospitals in the treatment ofhypoproteinemia, shock and other conditions requiring the use of aplasma expander. More recently, the value of such solutions has becomeenhanced by virtue of the increased risk of hepatitis associated withthe infusion of whole blood or plasma which has not been heattreated.

In most cases, solutions of plasma protein fractions are infused intothe patient at a slow rate and heretofore no side effects of anysignificance have been experienced with such solutions. Recently,however, solutions of plasma protein fractions have been used innumerous heart-lung bypass procedures for open-heart surgery where theinfusion rate is necessarily much greater, i.e., on the order of 100 ml.of 5 percent heattreated plasma protein solution within about fiveminutes. The arterial pressure has been observed to drop markedly in anumber of such patients whereas this undesirable effect could becontrolled if the infusion rates were reduced. Thus, the use of suchplasma solutions in situations requiring rapid infusion could beseriously detrimental to the patient when such a depressor effectoccurs.

It is an object of this invention to provide a method of removing thedepressor substance from heat-stable human plasma protein fractions.Another object is the provision of novel heat stable plasma protein,substantially free of blood pressure depressants. Other objects will beapparent to those skilled in the art to which this invention pertains.

SUMMARY OF THE INVENTION The depressor substance which causes a decreasein arterial pressure during the rapid infusion of solutions ofheat-treated plasma protein fractions, e.g., in heartlung by-passtechniques, can be substantially removed by contacting a solution of aheat-treated plasma protein fraction with a fractionating substance,including surface active adsorbents, cation exchangers, ultrafiltrationmembranes and gel filtration particles.

DETAILED DISCUSSION The starting materials for the process of thisinvention are stable human blood plasma protein fractions which containa depressor, i.e., a substance which lowers blood pressure significantlywhen plasma containing it is infused rapidly. Stable plasma proteinfractions are those which have been rendered heat stable by heating, upto 60 C., for up to hours. Such stable plasma protein fraction typicallyconsist predominantly of albumin plus small amounts of alpha and betaglobulins.

A preferred class of starting material is a nonhomogeneous plasmaprotein fraction, e.g., that obtained from Supernatant IV-l byprecipitation with ethanol (Cohn Method 6 process), which proteinfraction has been reconstituted to a 5 percent solution containing NaCland a stabilizer, e.g., acetyl tryptophan and/or sodium caprylate, andthen heated to 60 C. for 10 hours to destroy hepatitis virus. Thisstable plasma protein fraction is described in Japanese PatentPublication No. 5297/60 which issued as Japanese Pat. No. 265704, thecounterpart of U.S. Pat. No. 2,958,628. Another group of startingmaterials are solutions of plasma protein fractions which have beenheated at about 60 C. for shorter periods of time, e.g., between about 2to 10 hours, can also be used. For example, it has been observed thatheating solutions of stable plasma protein fractions at 60 C. for 10hours or longer to inactivate any hepatitis virus which may be present,sometimes causes a small amount of precipitate to form.

In a preferred aspect of this invention, a solution of a stable plasmaprotein fraction is heated at about 60 C. for a few hours, for example,about 1 to 4 hours. Any precipitate which forms is removed, e.g., byfiltration or centrifugation, and the clear plasma solution is thentreated with any one of the means of this invention for removingdepressor substances. Subsequently, heating at 60 C. for 10 hours orlonger may then be performed for the purpose of destroying any hepatitisvirus in the plasma. Another starting material is the heat-treatedstable plasma protein fraction obtained from human placenta.

The exact nature of the material employed for removing the depressor isnot critical and can readily be determined according to methods wellknown in the art, given the knowledge that the depressor is a relativelylow molecular weight, readily separable material. For example, using anultrafiltration membrane which allows materials of a molecular weightbelow 10,QO0 to pass through, the depressor can be separated from anystarting plasma protein solution. The operability of any fractionatingmaterial which is inert to plasma proteins 0 for separating thedepressor substance from the starting plasma solution can then bedeterminedusing this separated solution of the depressor to determineexperimentally the optimum conditions using that fractionating material.

It will also be apparent that once the depressor substance has beencharacterized, its removal from any starting plasma protein solution bya selected fractionating material can be readily determined by routineexperimentation.

Preferred fractionating materials for removing the depressor are surfaceactive adsorbents, cation exchangers, preferably cationic ion exchangeresins, ultrafiltration membranes and gel filtration molecular sieves,and accordingly, the preferred methods of removal of the depressor aresurface active adsorbent chromatography, cation exchangerchromatography, ultrafiltration and gel molecular sieve filtration.

A preferred treatment of the protein solution for removal of thedepressor substance or substances comprises treating the solution with asurface active adsorbent, for example, silica gel, hydrated alumina gel,magnesium hydroxide gel or barium sulfate, followed by separation of theadsorbent from the mixture to give a plasma protein solutionsubstantially free of depressor substances. Although more concentratedor less concentrated solutions of plasma protein can be used, aconcentration of about 5 percent is preferred. The

optimum amount of adsorbent used will vary, depending upon the particlesize of the adsorbent, the relative concentration of the protein, andthe desired level to which the depressor substance is to be reduced,i.e., either complete removal or substantially complete removal, i.e.,to a point where a slight depressor effect by rapid infusion of thesolution would not be harmful to the recipient. In general, silica gelhaving a particle size of about 10-40 microns is a preferred adsorbentalthough particle sizes greater or smaller than 10-40 microns areeffective. Typical of the preferred silica gels are Aerosil 200 (DegussaInc.) and Merck Silica Gel H.

The starting protein solution can be mixed with the adsorbent batchwisewith gentle agitation or it can be passed through a suitable column ofthe adsorbent. Treatment time is not critical, and, in fact, in thebatchwise procedure, mixing for several minutes or for several hoursappears to have substantially the same effect in the successful removalof the depressor substances. The temperature at which the operation isperformed also is not critical so long as it is maintained below thatwhich is detrimental to the plasma proteins and generally is in therange of about to about 60 C.

Following treatment of the protein solution with the adsorbent, thelatter can be removed by conventional means, e.g., centrifugation and/0rfiltration. The solution of plasma protein can then be sterile-filteredinto suitable containers and preferably then heated at about 60 C. forat least hours to inactivate any hepatitis virus that may be present, ifthe solution has not previously been subjected to prolonged heating.

Although surface active adsorbents are the preferred agents for theremoval of depressor substances from plasma proteins, other agents orconditions can be used to bring about the desired effect. Solutions ofstable plasma protein fractions can be treated with cation exchangers,for example, carboxymethyl cellulose; carboxymethyl Sephadex (PharmaciaFine Chemicals), which is a cross-linked dextran with terminalcarboxymethyl groups; Amberlite CG-50, which is a sulfonated polystyrenecross-linked with divinylbenzene sold by Rohm and Haas and Co.; andDowex 50-X2 which is a similar cation exchanger sold by Dow Chemical Co.The protein solutions may be allowed to pass through columns of the ionexchangers such as those previously equilibrated with 0.25 percentsodium chloride solution, at a rate of about 50-150 ml./hr./cm and at atemperature of about 4 to 60 C. The eluates are then substantially freeof depressor substances.

Another useful means for the removal of depressor substances from stableplasma protein fractions is by ultrafiltration. The starting proteinsolutions containing depressor substance can be subjected toultrafiltration using a suitable membrane of a porosity which allowspassage essentially only of low molecular weight species below about10,000 (which includes the depressor substance) under conditions whichare well known to those practicing ultrafiltration procedures.

One of a variety of suitable membranes is that described as UM10 Diafioultrafiltration membrane, which is a non-cellulosic polymer with ionicgroups on the surface, available from Amicon Corporation. Otheracceptable membranes are PMlO Diaflo membranes (also from Amicon),similar to UM10 membrane but nonionic; Pellicon type PSED (MilliporeCorporation) and Nitrocellulose membrane 8-1 2136 (Sartorius Division ofBrinkman Instruments).

The ultrafiltration procedure is well described in H. J. Bixler, R. W.Hausslein, L. M. Nelsen, Separation and purification of biologicalmaterials by ultrafiltration, Nat. Meeting Am. Inst. of Chem. Eng.,Cleveland, May, 1969; C. .1. Van Oss, P. M. Bronson, Removal of IgM fromserum by ultrafiltration, Anal. Biochem., 36, 464 (1970); D. Boutin, .1.Brodeur, Ultrafiltration of human serum, evidence of low-molecularweight cholinesterase activity (in French), Rev. Can. Biol., 29 (2), 187(June, 1970); Ultrafiltration for laboratory and clinical uses,Publication No. 403, 1970, put out by Amicon Corporation, Lexington,Mass.

Still another means for removal of depressor substances from stableplasma protein fractions according to the present invention is by gelfiltration. Similar in some respects to ultrafiltration, such a systemdepends on the ability of lower molecular weight species, including thedepressor substance, to pass through the interstices of bead-formed gelparticles and become more or less entrapped thereby. The procedure forthe use of molecular sieve gels in gel filtration is well known in theart and is exemplified in numerous references, including Whitaker, J.R.,Determination of molecular weights of proteins by gel filtration onSephadex, Anal. Chem., 35, 1950-1953 (1963); Andrews, P., Estimation ofthe molecular weights of proteins by Sephadex gel filtration, Biochem.,J., 91, 222-233 (1964); Laurent, T. C., Killander, J., A theory of gelfiltration and its experimental verification, J. Chromamg, 14, 317-330(1964); Carnegie, P. R., Estimation of molecular size of peptide by gelfiltration, Biochem. J., 95, 9 P (1965). Among those gels suitable foruse in the gel filtration method but not limited thereby to thesespecific examples are Sephadex G- 25, Sephadex G-50, Sephadex G andSephadex G- which are a variety of cross-linked dextran available fromPharmacia Fine Chemicals. Also useful is Sepharose 68, a bead-formedagarose gel available from Pharmacia.

The depressor substance which is removed from stable plasma proteinfractions is a polypeptide having a molecular weight between 1,000 and10,000. the depressor substance is believed to be generated primarilyduring the heating of the protein solution. However, we have found thatthis purified stable plasma protein fraction may thereafter be heated upto 60 C. for extended periods without generating additional depressorsubstance.

After contacting plasma protein fractions which contain this depressorsubstance with any one of the materials described herein for removingthe depressor substance according to the present invention, viz., asurface active adsorbent, a cation exchanger, an ultrafiltrationmembrane or by gel filtration particles, the resulting purified, stableplasma protein fraction, which is substantially free of depressorsubstance can be rapidly infused intravenously into a patient withoutthe danger of depressing the blood pressure, particularly in cases ofheart-lung by-pass. As a result of the present invention, a purifiedstable plasma protein fraction is obtained, whose scope of clinicalapplication is extended over that of previously obtainable plasmaprotein fractions.

The term substantially free of depressor as used herein means that theplasma protein fraction lacks true depressor activity, as distinguishedfrom the volume effect which causes a nominal drop in blood pressure,when any liquid, including saline or isotonic solution, is injected.This effect can be distinguished by the dog isolated hind limb test,which detects only true depressor effect. The novel depressor-freeplasma protein fraction products of this invention give a negativeresponse in this test and in the smooth muscle rat uterus test for kininand kinin-like substances.

It is believed that the'depressor substance may be bradykinin or akinin-like material, since it is well established that blood plasmacontains kininogens capable of being converted to kinins when activatedby a certain specific enzyme or enzymes. Treatment of plasma proteinfractions with either surface active adsorbents or cation exchangers orby ultrafiltration or gel filtration results in the removal of any kininor kinin-like substance, as evidenced by lack of significant depressoreffect when the treated protein is subjected to testing on smooth muscleor perfused in the isolated hind limb of a dog or administered bysystemic infusion in dogs. Contractions of the smooth muscle of a ratuterus is a highly sensitive test and quite specific for indicating thepresence of kinin or kinin-like substances. The isolated hind limb testis a highly sensitive test for detecting depressor substances whichcause dilation of the peripheral circulatory system thus producing afall in blood pressure. Systemic infusion is an in vivo test whichsimulates effects on blood pressure following rapid infusion in apatient.

For a description of techniques for the fractionation of human plasmaproteins with adsorbents, ion exchange resins, molecular sieve gelfiltration and ultrafiltration, see H. E. Schutze and J. F. Hermans,Molecular Biology of Human Proteins, Vol. I, pp. 285-303 (Elsevier.Pub., N.Y. 1966). For references to the removal of a kinin from bloodwith a cationic ion exchange resin, see M. E. Webster and J. P. Gilmore,Biochem. Pharm., Vol. 14, 1,161-l,163 (1965); J. A. Bates, L. Gillespieand D. T. Mason, The Lancet, Vol. 70, 514-517 (1964); and J. V. Pierceand M. E. Webster, Biochem. and Biophys. Res. Comm., Vol. 5, No. 5,353-357 (1961); ibid, with siliconized silica gel, H. Yoshida, K.Matsumoto, T. Nakajima and Z. Tamura, Chem. Pharm. Bull. 19(8)1,691-1,695 (1971).

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Starting Materials PlasmaProtein Fraction, Human A. From blood plasma, heat-treated at 60 C. forhours.

Most of the experimental studies were conducted on non-homogeneousplasma protein fractions obtained by the process described in US. Pat.No. 2,958,628. Various lots containing 5% protein were used which byelectrophoretic analysis contained at least 83 percent albumin and nomore than 17 percent of alpha globulin and beta globulin. In addition,the 5 percent protein solutions were stabilized with sodiumacetyltryptophanate (or N-acetyltryptophan) and sodium caprylate diumchloride in amounts which rendered the solution slightly less thanisotonic. The protein solutions had also been heated at about 60 C. for10 hours to destroy any hepatitis virus.

B. From blood plasma, heat-treated at 60 C. for 2 hours.

The dried plasma protein powder reconstituted to a 5 percent proteinsolution in water and stabilized with 0.004 M quantities of sodiumacetyltryptophanate and sodium caprylate, and also containing sodiumchloride in an amount to make the solution slightly less than isotonic(as described in US. Pat. No. 2,958,628) was heated at 60 C. for about 1to 4 hours, in this instance about 2 hours, rather than for 10 hours asin the case of Starting Material A. A small amount of flocculentprecipitate generally formed. After cooling to about room temperature,the precipitate was removed by filtration and the clear solution wasthen ready for use.

C. From placenta.

Human placentas which were frozen immediately after each delivery werefinely crushed. An extract from 20 kg. of this tissue with 40 liters of1 percent sodium chloride solution yielded a precipitate of gammaglobulin. The supernatant was mixed with butyric acid in amounts to makethe solution 2-6 percent with respect to the acid. The pH was adjustedto 4.5 5.5, the solution was heated at 57-60 C. for 1 to 2 hours, andthe precipitate which formed was removed. Ammonium sulfate was added tothe supernatant in amounts to provide a concentration of 35-40 percent.The precipitated protein was collected as paste and dialyzed againstrunning water at 4 C for 24 hours. The resulting product was anon-homogeneous plasma protein fraction comparable to Starting MaterialA, above. Its solutions in water were adjusted to about pH 7 foraltrafiltration, gel filtration or cation exchanger treatment.

Methods for Testing Depressor Substances A. Smooth MuscleContractibility.

Smooth muscle contractibility was measured by the method of Magnus(Trautshold, K., Handbook of Experimental Pharmacology, Vol. XXV, p. 55,Springer- Verlag, New York, 1970). A 1.5 cm. strip of uterus muscle wasisolated from a virgin Wistar rat weighing about 150 g. The strip wassuspended in 8.6 ml. de Jalon solution saturated with air and containing0.1 mg. percent of atropine sulfate. Kymograph recordings of thecontractile forces were made before and for seconds following theaddition of 0.4 ml. of the test solution.

B. Blood Pressure Following Systemic Infusion of Test Solution Male dogsof about 8 kg. body weight were anesthetized by intramuscular injectionof urethane (2.2 g./kg.), and the left carotid artery was cannulated forrecording arterial pressure with a polygraph transducer. The 5 percenttest solution was administered through a cannula inserted in the rightfemoral vein at a dose of -214 mg. of protein/kg. and at a rate of 18-30ml./minute. A depressor effect was expressed as a percentage decrease ofthe mean arterial pressure following infusion as compared to thepressure before infusion, i.e.,

Mean arterial pressure before infusion Mean arterial pressure afterinfusion 7: Decrease X 0 Mean arterial pressure before infusion 10 wayof polyethylene catheters at each end to the tied l artery, one catheterbeing placed well into the portion of the artery leadidng to the heartand the other catheter being placed about 6-10 cm. in the directionleading toward the limb extremity. A Sigma motor pump was connecteddirectly into the loop below the first named catheter and a pressuretransducer was connected into the loop on the peripheral side of thepump. Heparin (l0 mg./kg.) was injected I.V. and after 30 minutes, thepump was started and adjusted so that the pressure of the blood leavingthe pump to flow into the limb essentially matched the arterial pressurerecorded from the right femoral artery. Test solutions of percent plasmaprotein at a total dose of 250 mg. of pro-' tein (5 ml.) were infusedinto the artery of the isolated hind limb at a rate of l ml./5 seconds.A Sigma motor pump which provides a substantially constant pulsatileflow was used so that base lines in the pressure recordings could bedetermined more accurately. A base line is that figure obtained byadding to the diastolic pressure one-third of the difference betweenthesystolic and diastolic pressures. Decreases following the infusion ofthe test solutions were expressed as the actual difference in mm. Hg.between the baseline before infusion and the base line after infusion.

This isolated hind limb procedure is more sensitive than the systemicinfusion method (above) and will show direct effects of depressorsubstances on arterial pressure as a result of changes in peripheralresistance.

Removal of Depressor Substance A. Surface Active Adsorbent.

Example 1 A 5 percent solution of heat-treated plasma protein DepressorSubstance Method A Method B Starting Material A Strong Contraction" l1.5% Product of Example I No Contraction 0.0%

" Contraction was slightly greater than that obtained by 10 ng. ofBradykinin. Dose of I87 mg. protein/kg. at a rate of IS ml./min.

Silica gel effectively removed depressor substance from non-homogeneousplasma protein.

Example 2 100 ml. of heat-treated plasma protein solution (StartingMaterial A) was stirred gently at about 25 C. with 2.0 g. silica gel(Aerosil 200) for 5 hours, then centrifuged and the clear supernatantsolution was tested for depressor substance.

These results indicated the effectiveness of silica gel in a batchwisetreatment to remove depressor substance from plasma protein.

Example 3 100 ml. each of Starting Material A was stirred with 1.0 g.and 2.0 g. silica gel (Aerosil 200) for 4 hours at room temperature. Thetreated solutions were then tested for depressor substance.

. Depressor Substance Method C fraction (Starting Material A) was passedthrough a column of Merck H silica gel previously equilibrated with 0.25percent sodium chloride. The flow rate of the protein solution was -150ml./hr./cm About percent of the protein was recovered in the effluent asdetermined by optical density at 280 nm. in a Hitachi 6Spectrophotometer. By electrophoretic analysis, the final productcomprised 88.5 percent albumin, 7.5 percent alpha globulin and 4.0percent beta globulin.

The results show a ratio of 1 to 5 of silica gel to protein is about aseffective for removing depressor substance as a ratio of l to 2.5.

Example 4 A ml. portion of heat-treated plasma protein solution(Starting Material B) was stirred gently with 2 g. of silica gel(Aerosil 200) at room temperature for 3 hours. The silica gel wasremoved and a portion of the solution was heated at 60 C. for l 1 hours.

Depressor Substance Method A Method C Contraction. Av. No.

mm. Before After Decrease Detn.

Starting Material B l 1.5 166 I04 64 2 Silica gel treated product l I67139 28 2 Silica gel treated product (heated) 0 I67 I33 34 2 The resultsshow depressor substance iseffectively removed from plasma proteinheated for relatively short periods of time and that prolonged heatingof plasma protein which has been treated previously with silica gel toremove the depressor substance does not generate additional depressorsubstance.

Example Depressor Substance Method C Av. Decrease Starting Material AProduct of Example 5 The results show the effectiveness of aluminumhytion were combined. About 90 percent of the protein was recovered asdetermined by optical density at 280 nm. on this mixture.

Depressor Substance Method A Method B Starting Material C Strong LargeContraction Depression Product of Example 7 No Contraction No DepressionThe results show a weak cation exchanger effectively removes depressorsubstance from plasma protein.

Example 8 100 ml. of a 5 percent plasma protein solution StartingMaterial A) was stirred gently with 5 g. of moist Dowex 50-X2 (freshlyregenerated with dil. NaOH, then dil. HCl) for 3 hours at roomtemperature at pH 7.2. The resin was filtered off and the solutionshowed 85 percent recovery of protein as determined by optical densityat 280 nm.

Depressor Substance Method C droxide in removing depressor substancefrom plasma Before After o. Deter- Av. protein 7 4O Infusion Infusionmmations Decrease Starting Example 6 Material A I61 78 7 83 100 ml.portions of plasma protein solution (Starting gggr 8 I 61 HO 3 51Material A) were treated as follows with the indicated saline comm] 9 I16 results:

Silica Gel Method C Sample Adsorhent Amount Time Temp.. C. Av. DecreaseA Merck H 3 g. 45 min. 5 22 B Aerosil 200 2 g. 45 min. 5 25 C Aerosil200 2 g. 4 hours 25 Starting Material A The results show Merck H Silicagel to be as effective as Aerosil 200 in removing depressor substanceand that time or temperature do not materially affect the removal ofdepressor substance by silica gel.

B. Cation Exchanger Example 7 A 5 percent protein solution (StartingMaterial C) was allowed to pass through a column of carboxymethylSephadex which had previously been equilibrated with 0.05 M phosphatebuffer containing 0.1 M sodium chloride. The flow rate was between -150ml./hr./cm The column was then washed with the same buffer-salinesolution and the effluent and wash solu- The results show depressorsubstance is removed from plasma protein by treatment with a strongcation exchange resin.

C. Ultrafiltration Example 9 Depressor Substance Method A Method BStarting Material C Strong Contraction l9.l Product of Example 9 NoContraction 0.0

The results indicate ultrafiltration of plasma protein solutioneffectively removes depressor substance. The filtrate obtained in theabove experiment was tested by Method A and produced strongcontractions, indicating the depressor substance had a molecular weightbelow 10,000.

D. Gel Filtration Example 10 A column X 50 cm. filled with Sephadex G-50was charged with 70 ml. ofa plasma protein solution (Starting MaterialC) and eluted with 0.5 M sodium chloride solution at a rate of 4ml./min. Ten milliliter fractions were collected and assayed for proteincontent by optical density at 280 nm. The first 300 ml. of eluatecontained essentially all the protein. This 300 ml. portion wasconcentrated to 5 percent protein solution, additionalN-acetyl-tryptophan and sodium caprylate were added to bring therespective concentration of each to 0.004 M, and the solution was heatedat 60 C. for hours. When. tested by Method A, this final solution showedno depressor substance whereas the eluates collected from 580 to 730 ml.contained all the depressor substance. The Starting Material C producedstrong contractions of the uterine strip comparable to that produced by10 ng. of Bradykinin. This experiment demonstrates that depressorsubstance can be removed from plasma protein by gel filtration.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:

1. A sterile aqueous solution of an electrophoretically non-homogeneous,human, heat-stable plasma protein fraction suitable for rapidintravenous infusion, substantially free of depressor effects.

2. The solution of claim 1, stabilized with a stabilizer selected fromthe group consisting of sodium acetyltryptophanate, N-acetyltryptophanand sodium caprylate.

3. The solution of claim 1 wherein the protein concentration is about 5percent.

4. The solution of claim 3 wherein the protein comprises at least 83percent albumin and no more than 17 percent of alpha globulin and betaglobulin.

5. The solution of claim 1 wherein the plasma protein fraction consistsessentially of a mixture of 85-92 percent albumin, 4-10 percent globulinand 2-7 percent beta globulin.

6. The solution according to claim 5, heat-treated until free ofinfective hepatitis virus.

7. A process for the production of an electrophoreticallynon-homogeneous plasma protein solution which is substantially free ofdepressor substance so that the solution may be rapidly infusedintravenously without causing a significant fall in blood pressure,which comprises the step of contacting a solution of anelectrophoretically non-homogeneous human plasma protein containingdepressor substance with a material selected from the group consistingof surface active adsorbents, cation exchangers, ultrafiltrationmembranes and gel filtration molecular sieves which removes thedepressor substance from the protein. membranes and gel molecularsieves.

8. The process of claim 7 wherein the proteins of the starting proteinsolution are a mixture consisting of at least 83 percent albumin and notmore than 17 percent alpha-globulin and beta-globulin.

9. The process of claim 8 comprising the step of heating the startingstable protein solution prior to contacting the protein solution withthe material which removes the depressor.

10. The process of claim 9 wherein the heating step is conducted forabout 1 to 4 hours.

11. The process of claim 10 comprising the step of heating the proteinsolution after the removal of the depressor therefrom at about 60 C. fora period of time of at least 10 hours effective to destroy any hepatitisvirus therein.

12. The process of claim 7 wherein the depressor is removed bycontacting the starting solution of nonhomogeneous human plasma proteincontaining a depressor with a surface active adsorbent.

13. The process of claim 12 wherein the surface active adsorbent isselected from the group consisting of silica gel and aluminum hydroxidegel.

14. The process of claim 13 wherein the starting plasma solutioncontaining the depressor is a heattreated mixture of at least 83 percentalbumin and not more than 17 percent alpha-globulin and beta-globulin.

15. The process of claim 13 wherein the surface active adsorbent and thesolution containing the depressor are mixed in a batchwise operation.

16. The process of claim 13 wherein the plasma solution containing thedepressor substance is passed through a column of the surface activeadsorbent.

17. The process of claim 15 further including the step of separating thesurface active adsorbent from the mixture by centrifugation orfiltration.

18. The process of claim 7 wherein the depressor is removed bycontacting the starting solution of nonhomogeneous human plasma proteincontaining a depressor with a cation exchanger.

19. The process of claim 18 wherein the cation exchanger is a member ofthe group consisting of sulfonated polystyrene cross-linked withdivinylbenzene, carboxymethyl cellulose, and cross-linked dextran havingterminal carboxymethyl groups.

20. The process of claim 7 wherein the depressor is removed bycontacting the starting solution of nonhomogeneous human plasma proteincontaining a depressor with gel filtration molecular sieve.

21. The process of claim 20 wherein the gel filtration particles areselected from the group consisting of cross-linked dextrans and agarosegels having the capacity for entrapping substances with molecularweights below about 10,000.

22. The process of claim 7 wherein the depressor is removed by filteringthe starting solution of nonhomogeneous human plasma protein containinga depressor with an ultrafiltration membrane which will allow thedepressor substance to pass through the membrane into thefiltrate butprevents the passage of substantially all of the desired plasmaproteins.

23. The process of claim 7 which comprises the steps of a. heating asolution of a non-homogeneous stable human plasma protein fraction atabout 60 C.,

b. contacting the heated solution with a material selected from thegroup consisting of surface active adsorbents, cation exchangers,ultrafiltration membranes and gel filtration molecular sieves.

24. The process of claim 23 comprising the step of cooling the solutionprior to step b).

25. The process of claim 24 comprising the step of separating anyprecipitate from the cooled solution.

26. The process of claim 25 comprising heating the solution in step a)for about 1 to 4 hours and again heating the cooled solution, for about10 hours at about 60 C.

27. The process of claim 26 wherein in Step b) the material is a surfaceactive agent selected from the group consisting of silica gel andaluminum hydroxide gel.

UNITED STATES PATENT OFFICE CERTIFICATE OF CU ECTIN PATENT N0. 3 37 ,775

DATED April 8, 1975 INVENTOR(S) lzaka et a1 It is certified that errorappears in the above-identified patent and that said Letters Patent arehereby corrected as shown beiow:

Column 4, line 41, change "the" to --The--.

Column 7,'line 12, change "leadidng" to --leading.

Column 11, line 64, change "4-10 percent globulin" to --4-10 percentalpha globulin- Column 12, lines 12 and 13, after the period, delete"membranes and gel molecular sieves."

Signed and sealed this 15th day of July 1975.

(SEAL) Attest:

C. MARSHALL DANN RUTH ,C. MASON Commissioner of Patents AttestingOfficer and Trademarks

1. A STERILE AQUEOUS SOLUTION OF AN ELECTROPHORETICALLY NONHOMOGENOUS,HUMAN, HEAT-STABLE PLASMA PROTEIN FRACTION SUITABLE FOR RAPIDINTRAVENOUS INFUSION, SUBSTANTIALLY FREE OF DEPR3ESSOR EFFECTS.
 2. Thesolution of claim 1, stabilized with a stabilizer selected from thegroup consisting of sodium acetyltryptophanate, N-acetyltryptophan andsodium caprylate.
 3. The solution of claim 1 wherein the proteinconcentration is about 5 percent.
 4. The solution of claim 3 wherein theprotein comprises at least 83 percent albumin and no more than 17percent of alpha globulin and beta globulin.
 5. The solution of claim 1wherein the plasma protein fraction consists essentially of a mixture of85-92 percent albumin, 4-10 percent globulin and 2-7 percent betaglobulin.
 6. The solution according to claim 5, heat-treated until freeof infective hepatitis virus.
 7. A process for the production of anelectrophoretically non-homogeneous plasma protein solution which issubstantially free of depressor substance so that the solution may berapidly infused intravenously without causing a significant fall inblood pressure, which comprises the step of contacting a solution of anelectrophoretically non-homogeneous human plasma protein containingdepressor substance with a material selected from the group consistingof surface active adsorbents, cation exchangers, ultrafiltrationmembranes and gel filtration molecular sieves which removes thedepressor substance from the protein. membranes and gel molecularsieves.
 8. The process of claim 7 wherein the proteins of the startingprotein solution are a mixture consisting of at least 83 percent albuminand not more than 17 percent alpha-globulin and beta-globulin.
 9. Theprocess of claim 8 comprising the step of heating the starting stableprotein solution prior to contacting the protein solution with thematerial which removes the depressor.
 10. The process of claim 9 whereinthe heating step is conducted for about 1 to 4 hours.
 11. The process ofclaim 10 comprising the step of heating the protein solution after theremoval of the depressor therefrom at about 60.degree. C. for a periodof time of at least 10 hours effective to destroy any hepatitis virustherein.
 12. The process of claim 7 wherein the depressor is removed bycontacting the starting solution of non-homogeneous human plasma proteincontaining a depressor with a surface active adsorbent.
 13. The processof claim 12 wherein the surface active adsorbent is selected from thegroup consisting of silica gel and aluminum hydroxide gel.
 14. Theprocess of claim 13 wherein the starting plasma solution containing thedepressor is a heat-treated mixture of at least 83 percent albumin andnot more than 17 percent alpha-globulin and beta-globulin.
 15. Theprocess of claim 13 wherein the surface active adsorbent and thesolution containing the depressor are mixed in a batchwise operation.16. The process of claim 13 wherein the plasma solution containing thedepressor substance is passed through a column of the surface activeadsorbent.
 17. The process of claim 15 further including the step ofseparating the surface active adsorbent from the mixture bycentrifugation or filtration.
 18. The process of claim 7 wherein thedepressor is removed by contacting the starting solution ofnon-homogeneous human plasma protein containing a depressor with acation exchanger.
 19. The process of claim 18 wherein the cationexchanger is a member of the group consisting of sulfonated polystyrenecross-linked with divinylbenzene, carboxymethyl cellulose, andcross-linked dextran having terminal carboxymethyl groups.
 20. Theprocess of claim 7 wherein the depressor is removed by contacting thestarting solution of non-homogeneous human plasma protein containing adepressor with gel filtration molecular sieve.
 21. The process of claim20 wherein the gel filtration particles are selected from the groupconsisting of cross-linked dextrans and agarose gels having the capacityfor entrapping substances with molecular weights below about 10,000. 22.The process of claim 7 wherein the depressor is removed by filtering thestarting solution of non-homogeneous human plasma protein containing adepressor with an ultrafiltration membrane which will allow thedepressor substance to pass through the membrane into the filtrate butprevents the passage of substantially all of the desired plasmaproteins.
 23. The process of claim 7 which comprises the steps of a.heating a solution of a non-homogeneous stable human plasma proteinfraction at about 60.degree. C., b. contacting the heated solution witha material selected from the group consisting of surface activeadsorbents, cation exchangers, ultrafiltration membranes and gelfiltration molecular sieves.
 24. The process of claim 23 comprising thestep of cooling the solution prior to step b).
 25. The process of claim24 comprising the step of separating any precipitate from the cooledsolution.
 26. The process of claim 25 comprising heating the solution instep a) for about 1 to 4 hours and again heating the cooled solution,for about 10 hours at about 60.degree. C.
 27. The process of claim 26wherein in Step b) the material is a surface active agent selected fromthe group consisting of silica gel and aluminum hydroxide gel.